| Malolactic fermentation(MLF) plays very important role in winemaking. MLF can not only decrease total wine acidity and improve the taste, but also increase the biological stability of wine and bring some beneficial aroma for wine. However, limited knowledge has been known by people until now. A number of key questions remain, in particular some methods and technologies to study the malolactic bacteria has not been established, which made the research about malolactic bacteria hysteretic. In this study, we established the methods of identification and genotyping by studing the malolactic bacteria isolated from Chinese and Australian wine region. Genetic polymorphism, ethanol tolerance and quick method of counting bacteria cells by flow cytometry also were studied for the purpose of further understanding MLF and malolactic bacteria.In order to understand the genetic polymorphism of O. oeni, amplify fragment length polymorphism(AFLP) of Hind III and Mse I digests of the genomic DNA of the O. oeni strains was developed for the first time to genotype the strains. DNA extraction, digestion and ligation, pre-selective and selective amplification methods were developed. Denaturing polyacrylamide gel electrophoresis and capillary electrophoresis were used to detect the selective PCR product respectively. To assess the accuracy of data analysis, two softwares were used for analyzing the data from capillary electrophoresis. Primer combinations were selected for better amplification efficiency. The results showed that AFLP with Hind III and Mse I was successfully able to reproducibly(>98%) genotype O. oeni, HT-MA, HT-MT, HT-MC, HG-MA, HG-MT, HC-MT were showed the best primer combinations.AFLP was used to analyze 22 bacteria strains which isolated from different Chinese wine regions. Cluster analysis indicated that the 22 O. oeni strains were fall into three group, and the genetic similarity between these strains is low, not all strains from same region have high genetic similarity, those results indicated that the O. oeni strains isolated from China have very rich genetic diversity and the genetic similarity of O. oeni strains not only related to their ecological geographic distribution, maybe also micro-ecological environment.Alcoholic fermentation(AF) and MLF assays were carried out in Australian Grenache grapes which contained potentially high ethanol. The results showed that all the uninoculate AF wines can finish MLF, but only 1/3 of the inoculate AF wines can finish MLF. 108 lactic acid bacteria isolates were obtained from spontaneous MLF. Morphological observations, gram staining, catalase test, 16 S r RNA and species-specific PCR showed that 104 of these were O. oeni, three were L. hilgardii, one was Staphylococcus pasteuri(S. pasteuri).AFLP analysis of 104 O. oeni strains isolated from Australian wines indicated that they were contained within six different principal clusters, and their similarity level was low(0.570~0.704), that means the diversity of the 104 O. oeni strains is rich.In order to find a quick method to count O. oeni and L. hilgardii cells, Bac LightTM kit were used to Staining bacteria cells and flow cytometry(FCM) were used to detect the staining cells. The FCM counting results of L. hilgardii were correlated with plate counting, but not the O. oeni strains.Based on the cluster analysis, ten O. oeni strains were chosen for carrying out an ethanol tolerance assay. Several O. oeni strains(G63, G46, G71 and G39) survived and grew well in MRS-AJ with 17%(v/v) ethanol, while the commercial O. oeni reference strain did not. Strain G63 could also survive and grow more than 168 hours after inoculated in MRS-AJ medium with 19%(v/v) ethanol. These results suggest that O. oeni G63, G46, G71 and G39 could potentially be used as MLF starters for high ethanol content wines. All three L. hilgardii strains could survive and grow in MRS-AJ with 19%(v/v) ethanol, perhaps also indicating their suitability as next generation MLF starter cultures. |
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